Immunocytochemistry analysis of 293T cells starved for 24 hours then treated with 100nM insulin for 30 minutes labeling Phospho-eIF4EBP1 (S65) with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (HA723106) at 1/1,000 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (HA723106) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Phospho-eIF4EBP1 (S65) was immunoprecipitated from 0.2 mg 293T cell lysate with HA723106 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723106 at 1/1,000 dilution. Alpaca anti-Rabbit IgG Fc secondary antibody (HA1031) at 1/50,000 dilution was used for 1 hour at room temperature. Lane 1: 293T cell lysate (input) Lane 2: HA723106 IP in 293T cell lysate Lane 3: Rabbit IgG instead of HA723106 in 293T cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 18 seconds; ECL: K1801